甲基-CpG结合结构域蛋白2抑制PRRSV复制的研究  

Studies on Methyl-CpG binding domain protein 2(MBD2) inhibiting the replication of PRRSV

作　　者：赵慧君 赵旭阳 史西保[2,3] 陈静 范小敏[3] 王丽 李青梅[2] 常晓博[2,4] 邓瑞广 张改平[1,2] ZHAO Huijun;ZHAO Xuyang;SHI Xibao;CHEN Jing;FAN Xiaomin;WANG Li;LI Qingmei;CHANG Xiaobo;DENG Ruiguang;ZHANG Gaiping(College of Life Sciences,Henan Agricultural University,Zhengzhou 450002,China;Henan Provincial Key Laboratory of Animal Immunology,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China;College of Life Sciences,Henan Normal University,Xinxiang 453002,China;College of Veterinary Medicine,Northwest A&F University,Yangling 712100,China)

机构地区：[1]河南农业大学生命科学学院,河南郑州450002 [2]河南省农业科学院动物免疫学重点实验室,河南郑州450002 [3]河南师范大学生命科学学院,河南新乡453002 [4]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《河南农业大学学报》2019年第6期903-911,917,共10页Journal of Henan Agricultural University

基　　金：河南省自然科学基金项目(182300410077);农业部动物免疫学重点实验室、河南省动物免疫学重点实验室开放课题(PKLAI20170605);河南省高等学校重点科研项目(17A180006);河南师范大学优秀青年科学基金项目(校20170031);2018年河南师范大学大学生创新创业训练项目

摘　　要：为了研究甲基-CpG结合结构域蛋白2(MBD2)在体外对猪繁殖与呼吸综合征病毒(PRRSV)的复制是否有抑制作用。首先,构建MBD2的3’-UTR报告质粒,通过双荧光素酶报告结果表明MBD2是miR-373靶基因。然后用MOI=1的PRRSV感染MARC-145细胞,在24、36、48 h后分别取样,实时荧光定量PCR(qRT-PCR)和Western blots试验进行检测。结果表明,PRRSV感染下调MBD2的mRNA和蛋白的表达水平。用真核表达载体pcDNA3.1-Flag构建了MBD2的真核表达质粒pcDNA3.1-Flag-MBD2,用pcDNA3.1-Flag-MBD2转染MARC-145细胞,24 h后感染PRRSV,48 h后收获细胞。TCID50的试验结果表明,过表达MBD2能降低PRRSV滴度,而qRT-PCR和Western blots的结果则表明MBD2能降低PRRSV的载量;相反,MBD2的siRNA试验表明,下调表达有利于PRRSV在MARC-145细胞复制。通过基因缺失试验,构建MBD2部分结构域缺失的表达质粒,最终发现MBD2的GR与MBD结构域可能在MBD2抑制PRRSV复制起关键的作用。研究结果表明,MBD2是拮抗PRRSV的宿主内源性蛋白,并发现MBD2的GR与MBD结构域可能在MBD2抑制PRRSV复制中起关键的作用,而PRRSV可能利用miR-373来下调MBD2的表达,从而逃逸宿主对病毒的清除。Studies were conducted to investigate whether methyl-CpG binding domain protein 2(MBD2)inhibits the replication of porcine reproductive and respiratory syndrome virus(PRRSV)in vitro.Firstly,the 3′-UTR reporter plasmid of MBD2 was constructed,and the results of the dual luciferase reporter experiment showed that MBD2 was the target gene of miR-373.Then,PRRSV with MOI=1 was used to infect Marc-145 cells.Samples were taken 24,36 and 48 h later,and the results of qRT-PCR and Western blots showed that PRRSV infection down-regulated the mRNA and protein expression levels of MBD2.The eukaryotic expression plasmid pcDNA3.1-Flag-MBD2 was constructed by using the eukaryotic expression vector pcDNA3.1-Flag.Marc-145 cells were transfected with pcDNA3.1-Flag-MBD2,then infected with PRRSV 24h later and harvested 48 h later.The TCID50 results showed that over-expression of MBD2 could reduce the PRRSV titer,while the results of qRTPCR and Western blots showed that over-expression of MBD2 could reduce the PRRSV load.On the contrary,the siRNA assay showed that the down-regulated expression of MBD2 was beneficial to PRRSV replication in Marc-145 cells.Finally,expression plasmids with partial domain deletion of MBD2 were constructed through gene deletion experiments,and it was found that the GR and MBD domain of MBD2 may play a key role in the inhibition of PRRSV replication.The research results show that MBD2 is an endogenous host protein antagonizing PRRSV,and that GR and MBD domain of MBD2 may play a key role in inhibiting PRRSV replication,while PRRSV may use miR-373 to downregulate MBD2 expression,thus escaping from the host to clear the virus.

关 键 词：猪繁殖与呼吸综合征病毒 甲基-CpG结合结构域蛋白2 miR-373 

分 类 号：S855[农业科学—临床兽医学]